Biosensors are devices that sense analytes of interest using biometric materials and convert the results into physicochemical signals. Useful biosensors should be selective for particular substances, sensitive to small amounts of analytes, able to respond in a short time, and inexpensive. These requirements determine the performance of biosensors and can increase the competitiveness of biosensors in the market. Many challenges and fierce competition are underway around the world to overcome the functional limitations of existing biosensors while meeting the above requirements. The medical field accounts for about half of the entire biosensor market. Portable, compact, and simple biosensors are emerging in the market that can be used wherever and whenever necessary.
Due to excellent characteristics of biosensors over conventional analyzers, efforts have been dedicated to developing efficient biosensors capable of replacing time-, labor-, and cost-consuming conventional testing methods. Various types of biosensor developed hitherto have been used in various industrial applications, such as medical, environmental, military, and automotive applications. Particularly, the medical diagnostic field is the biggest market of biosensors. Most biosensors have been developed to analyze low-molecular-weight substances, such as internal metabolites (e.g., saccharides), using enzymes as recognition elements and analyze structurally complex substances, such as hormones, proteins, and cells, using specific recognition elements, such as antibodies. Biosensors are of increasing interest in food inspection and safety applications. Under these circumstances, there has been much research aimed at developing biosensors. Biosensors are useful in analyzing not only food constituents, such as carbohydrates, proteins, and fats, but also low-abundant ingredients, such as cholesterol, alcohols, and vitamins. Furthermore, biosensors have been considered promising sensors for detecting harmful substances derived from foods. Biosensors can detect chemicals, such as heavy metals, antibiotics, and agrochemical residues, as well as biological substances, such as microbes.
Ion-selective electrodes (ISEs) are mainly used to detect the presence of polyvalent metal ions (Ca2+, Fe2+, Fe3+, Zn2+, Mn2+, Mg2+, Cu2+, etc.) in low-molecular-weight substances. For example, Korean Patent Publication No. 1997-0010882 A discloses an ion-selective electrode including an electrode and a conjugate membrane applied to the electrode wherein a particular metal ion can be selectively attached to the conjugate membrane. However, such conventional ion measurement techniques suffer from the occurrence of false positives and increased measurement variations, leading to poor analytical performance, in the case of analyte samples composed of very complex matrices, such as blood samples. Thus, biosensors using biometric materials need to be developed.
Calcium is the fifth most abundant element in the body and is present in an amount of about 1000 g in the adult body. Calcium is an essential nutrient that is available only through food intake. Of the calcium in the adult body, 99% is distributed in bones and teeth, and the rest is present in blood and soft tissues such as cartilage and muscle fibers. Blood calcium exists in various forms: combined with plasma proteins (40%), interacted in complex manner with anions (e.g., carbonic acid, lactic acid, and fluorophoreic acid; 10%), or liberated as free Ca2+ (50%). The free calcium level is about 1.25 mmol/L. Among the different forms of blood calcium, the balance of which is normally maintained, the free ionic portion is physiologically activated only during illness. Blood calcium ion plays a crucial role in blood coagulation and acts as a cofactor for diverse enzymes. Calcium ion is involved in physiological functions such as muscular contraction, neurotransmission, hormone secretion, and intracellular metabolism. Plasma calcium ion level in healthy humans is about 1.1-1.3 mmol/L. Patients whose calcium ion level is ≥1.3 mmol/L are diagnosed as having hypercalcemia and patients whose calcium ion level is ≤1.15 mmol/L are diagnosed as having hypocalcemia. A sudden change in blood calcium ion level adversely affects the body and is a life-threatening signal in severe cases.
A change in blood calcium ion level needs to be monitored and the importance of calcium-containing food intake is being emphasized, leading to the development of various methods for calcium concentration measurement. Current representative methods for the measurement of calcium concentration include chemical, atomic absorption spectroscopy, and chromophore-based spectrophotometry methods, in addition to methods based on the use of ion-selective electrodes (Ms). The most traditional chemical and atomic absorption spectroscopy methods have high accuracy but are disadvantageous in that pretreatment procedures and expensive instruments are required. Electrochemical methods using ion-selective electrodes and chromophore-based spectrophotometry methods have received attention due to their relative simplicity. However, the electrochemical methods have large measurement variations depending on sample composition and lack reproducibility. Another disadvantage of the electrochemical methods is that reagents are difficult to store. Chromophore-based spectrophotometry methods are less selective due to cross-reactivities with other ions.
Furthermore, since the measurement of blood ionic calcium level is greatly affected by blood pH and other environmental conditions, direct measurement after blood collection is most preferred. However, it is difficult to store blood samples isolated from individual patients in large hospitals in view of current medical systems. Further, on-site diagnostic equipment based on ion-selective measurement of ionic calcium is unsuitable for reagent storage and is expensive. For these reasons, large hospitals employ chromophore-based spectrophotometry methods to measure the calcium levels of general patients other than newborns and patients in the intensive care unit. Chromophore-based spectrophotometry methods enable blood storage and can be performed at reduced cost in the central laboratory. Blood calcium levels are calculated from the measured total calcium levels through various algorisms and the calculated values are used for medical diagnosis. As explained above, however, the compensation algorisms are always exposed to problems and limitations and do not perfectly reflect exact calcium ion levels of individual patients.
Current medical diagnostic situations associated with the measurement of polyvalent metal ions (Ca2+, Fe2+, Fe3+, Zn2+, Mn2+, Mg2+, Cu2+, etc.) similarly apply to veterinary diagnosis. Particularly, there is an increasing need to measure polyvalent metal ions in the field of food inspection but satisfactory alternatives are not yet available. Since ingredients present in most foods affect human health when ingested or otherwise taken into the body, it is necessary to quantitatively analyze food constituents using various assay techniques. Calcium ion is a typical example of polyvalent metal ions in food ingredients that need to be quantitatively analyzed. Calcium is the most abundant mineral in the body despite its relatively small amount circulating in blood. As explained above, blood calcium ions play a crucial role in regulating and controlling intracellular and extracellular physiological processes. The incidence of disease can be predicted depending on blood calcium ion level. Blood calcium ion level is even involved in the prevention of some cancers or osteoporosis. The elderly are at risk of calcium deficiency. In an effort to reduce this risk, calcium should be supplied through food intake. Currently, the consumption of calcium-containing foods is steadily on the rise.
The above calcium quantification methods can be applied to food samples. However, most of the methods require sample pretreatment, making it difficult to quickly inspect foods or automate periodic analysis processes during food production or management. Particular minerals in food are quantified by chemical methods or atomic absorption spectroscopy methods for quantitative analysis of polyvalent metal ions (Ca2+, Fe2+, Fe3+, Zn2+, Mn2+, Mg2+, Cu2+, etc.) after sample pretreatment for metal ion chelation and additional processing for measuring chelated conjugates. In contrast, ion selective electrodes (ISEs) have attracted much attention because of their potential to reduce the number of analysis processes and relative low price. However, currently commercially available ISEs have the problems of low analytical accuracy and poor stability when used to analyze complex samples, such as blood and food samples.